Since the discovery of penicillin, the use of antibiotics to treat the ravages of bacterial infections has saved millions of lives. With the advent of these xe2x80x9cmiracle drugs,xe2x80x9d for a time it was popularly believed that humanity might, once and for all, be saved from the scourge of bacterial infections. In fact, during the 1980s and early 1990s, many large pharmaceutical companies cut back or eliminated antibiotics research and development. They believed that infectious disease caused by bacteria finally had been conquered and that markets for new drugs were limited. Unfortunately, this belief was overly optimistic.
The tide is beginning to turn in favor of the bacteria as reports of drug resistant bacteria become more frequent. The United States Centers for Disease Control announced that one of the most powerful known antibiotics, vancomycin, was unable to treat an infection of the common Staphylococcus aureus (staph). This organism is commonly found in our environment and is responsible for many nosocomial infections. The import of this announcement becomes clear when one considers that vancomycin was used for years to treat infections caused by stubborn strains of bacteria, like staph. In short, the bacteria are becoming resistant to our most powerful antibiotics. If this trend continues, it is conceivable that we will return to a time when what are presently considered minor bacterial infections are fatal diseases.
There are a number of causes for the predicament in which practitioners of medical arts find themselves. Over-prescription and improper prescription habits by some physicians have caused an indiscriminate increase in the availability of antibiotics to the public. The patient is also partly responsible, for even in instances where an antibiotic is the appropriate treatment, patients will often improperly use the drug, the result being yet another population of bacteria that is resistant, in whole or in part, to traditional antibiotics.
The bacterial scourges that have haunted humanity remain, in spite of the development of modem scientific practices to deal with the diseases that they cause. Drug resistant bacteria are now advancing on the health of humanity. A new generation of antibiotics to once again deal with the pending health threat that bacteria present is required.
As more and more bacterial strains become resistant to the panel of available antibiotics, new compounds are required. In the past, practitioners of pharmacology would have to rely upon traditional methods of drug discovery to generate novel, safe and efficacious compounds for the treatment of disease. Traditional drug discovery methods involve blindly testing potential drug candidate-molecules, often selected at random, in the hope that one might prove to be an effective treatment for some disease. The process is painstaking and laborious, with no guarantee of success. Today, the average cost to discover and develop a new drug is nearly US $500 million, and the average time is 15 years from laboratory to patient. Improving this process, even incrementally, would represent a huge advance in the generation of novel antimicrobial agents.
Newly emerging practices in drug discovery utilize a number of biochemical techniques to provide for directed approaches to creating new drugs, rather than discovering them at random. For example, gene sequences and proteins encoded thereby that are required for the proliferation of an organism make for excellent targets since exposure of bacteria to compounds active against these targets would result in the inactivation of the organism. Once a target is identified, biochemical analysis of that target can be used to discover or to design molecules that interact with and alter the functions of the target. Using physical and computational techniques, to analyze structural and biochemical targets in order to derive compounds that interact with a target is called rational drug design and offers great future potential. Thus, emerging drug discovery practices use molecular modeling techniques, combinatorial chemistry approaches, and other means to produce and screen and/or design large numbers of candidate compounds.
Nevertheless, while this approach to drug discovery is clearly the way of the future, problems remain. For example, the initial step of identifying molecular targets for investigation can be an extremely time consuming task. It may also be difficult to design molecules that interact with the target by using computer modeling techniques. Furthermore, in cases where the function of the target is not known or is poorly understood, it may be difficult to design assays to detect molecules that interact with and alter the functions of the target. To improve the rate of novel drug discovery and development, methods of identifying important molecular targets in pathogenic microorganisms and methods for identifying molecules that interact with and alter the functions of such molecular targets are urgently required.
Escherichia coli represents an excellent model system to understand bacterial biochemistry and physiology. The estimated 4288 genes scattered along the 4.6xc3x97106 base pairs of the Escherichia coli (E. coli) chromosome offer tremendous promise for the understanding of bacterial biochemical processes. In turn, this knowledge will assist in the development of new tools for the diagnosis and treatment of bacteria-caused human disease. The entire E. coli genome has been sequenced, and this body of information holds a tremendous potential for application to the discovery and development of new antibiotic compounds. Yet, in spite of this accomplishment, the general functions or roles of many of these genes are still unknown. For example, the total number of proliferation-required genes contained within the E. coli genome is unknown, but has been variously estimated at around 200 to 700 (Armstrong, K. A. and Fan, D. P. Essential Genes in the metB-malB Region of Escherichia coli K12, 1975, J. Bacteriol. 126: 48-55).
Novel, safe and effective antimicrobial compounds are needed in view of the rapid rise of antibiotic resistant microorganisms. However, prior to this invention, the characterization of even a single bacterial gene was a painstaking process, requiring years of effort. Accordingly, there is an urgent need for more novel methods to identify and characterize bacterial genomic sequences that encode gene products required for proliferation and for methods to identify molecules that interact with and alter the functions of such genes and gene products.
One embodiment of the present invention is a purified or isolated nucleic acid sequence consisting essentially of one of SEQ ID NOs: 1-127, wherein expression of said nucleic acid inhibits proliferation of a microorganism. The nucleic acid sequence may be complementary to at least a portion of a coding sequence of a gene whose expression is required for proliferation of a microorganism. The nucleic acid sequence may be complementary to at least a portion of an RNA required for proliferation of a microorganism. The RNA may be an RNA encoding more than one gene product.
Another embodiment of the present invention is a nucleic acid comprising a fragment of one of SEQ ID NOs.: 1-127, said fragment selected from the group consisting of fragments comprising at least 10, at least 20, at least 25, at least 30, at least 50 and more than 50 consecutive bases of one of SEQ ID NOs: 1-127.
Another embodiment of the present invention is a vector comprising a promoter operably linked to the nucleic acid sequences of each of the preceding paragraphs. The promoter may be active in a microorganism selected from the group consisting of Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Saccharomyces cerevisae, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum, and Yersinia pestis or any species falling within the genera of any of the above species.
Another embodiment of the present invention is a host cell containing the vectors of the preceding paragraph.
Another embodiment of the present invention is a purified or isolated nucleic acid consisting essentially of the coding sequence of one of SEQ ID NOs: 128-298.
Another embodiment of the present invention is a fragment of the nucleic acid of the preceding paragraph, said fragment comprising at least 10, at least 20, at least 25, at least 30, at least 50 or more than 50 consecutive bases of one of SEQ ID NOs: 128-298.
Another embodiment of the present invention is a vector comprising a promoter operably linked to the nucleic acid of the preceding two paragraphs.
Another embodiment of the present invention is a purified or isolated antisense nucleic acid comprising a nucleic acid sequence complementary to at least a portion of an intragenic sequence, intergenic sequence, sequences spanning at least a portion of two or more genes, 5xe2x80x2 noncoding region, or 3xe2x80x2 noncoding region within an operon comprising a proliferation-required gene whose activity or expression is inhibited by an antisense nucleic acid comprising one of SEQ ID NOs.: 1-127.
Another embodiment of the present invention is a purified or isolated nucleic acid comprising a nucleic acid having at least 70% identity to a sequence selected from the group consisting of SEQ ID NOs.: 1-127, fragments comprising at least 25 consecutive nucleotides of SEQ ID NOs.: 1-127, the sequences complementary to SEQ ID NOs.: 1-127 and the sequences complementary to fragments comprising at least 25 consecutive nucleotides of SEQ ID NOs.: 1-127 as determined using BLASTN version 2.0 with the default parameters. The nucleic acid may be from an organism selected from the group consisting of Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, Enterobacter cloacae, Enterococcus faecalis, Staphylococcus aureus, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Saccharomyces cerevisae, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum, and Yersinia pestis or any species falling within the genera of any of the above species.
Another embodiment of the present invention is a vector comprising a promoter operably linked to a nucleic acid encoding a polypeptide whose expression is inhibited by an antisense nucleic acid comprising one of SEQ ID NOs.: 1-127. The polypeptide may comprise a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 299-469.
Another embodiment of the present invention is a host cell containing the vector of the preceding paragraph.
Another embodiment of the present invention is a purified or isolated polypeptide comprising a polypeptide whose expression is inhibited by an antisense nucleic acid comprising one of SEQ ID NOs.: 1-127, or a fragment selected from the group consisting of fragments comprising at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 or more than 60 consecutive amino acids of one of the said polypeptides. The polypeptide may comprise a polypeptide comprising one of SEQ ID NOs.: 299-469 or a fragment comprising at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 or more than 60 consecutive amino acids of a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 299-469.
Another embodiment of the present invention is a purified or isolated polypeptide comprising a polypeptide having at least 25% identity to a polypeptide whose expression is inhibited by a sequence selected from the group consisting of SEQ ID NOs.: 1-127, or at least 25% identity to a fragment comprising at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 or more than 60 consecutive amino acids of a polypeptide whose expression is inhibited by a nucleic acid selected from the group consisting of SEQ ID NOs.: 1-127 as determined using FASTA version 3.0t78 with the default parameters. The polypeptide may have at least 25% identity to a polypeptide comprising one of SEQ ID NOs: 299-469 or at least 25% identity to a fragment comprising at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 or more than 60 consecutive amino acids of a polypeptide comprising one of SEQ ID NOs.: 299-469 as determined using FASTA version 3.0t78 with the default parameters.
Another embodiment of the present invention is an antibody capable of specifically binding one of the polypeptides of the preceding paragraph.
Another embodiment of the present invention is a method of producing a polypeptide, comprising introducing a vector comprising a promoter operably linked to a nucleic acid encoding a polypeptide whose expression is inhibited by an antisense nucleic acid comprising one of SEQ ID NOs.: 1-127 into a cell. The method may further comprise the step of isolating said polypeptide. The polypeptide may comprise a sequence selected from the group consisting of SEQ ID NOs.: 299-469.
Another embodiment of the present invention is a method of inhibiting proliferation of a microorganism comprising inhibiting the activity or reducing the amount of a gene product whose expression is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127 or inhibiting the activity or reducing the amount of a nucleic acid encoding said gene product. The gene product may comprise a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 299-469.
Another embodiment of the present invention is a method for identifying a compound which influences the activity of a gene product required for proliferation, said gene product comprising a gene product whose expression is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127, said method comprising contacting said gene product with a candidate compound and determining whether said compound influences the activity of said gene product. The gene product may be a polypeptide and said activity may be an enzymatic activity. The gene product may be a polypeptide and said activity may be a carbon compound catabolism activity. The gene product may be a polypeptide and said activity may be a biosynthetic activity. The gene product may be a polypeptide and said activity may be a transporter activity. The gene product may be a polypeptide and said activity may be a transcriptional activity. The gene product may be a polypeptide and said activity may be a DNA replication activity. The gene product may be a polypeptide and said activity my be a cell division activity. The gene product may be a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 299-469.
Another embodiment of the present invention is a compound identified using the methods of the preceding paragraph.
AThe method of claim 28, wherein said gene product is a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 299-469. method for identifying a compound or nucleic acid having the ability to reduce the activity or level of a gene product required for proliferation, said gene product comprising a gene product whose activity or expression is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127, said method comprising:
(a) providing a target that is a gene or RNA, wherein said target comprises a nucleic acid encoding said gene product;
(b) contacting said target with a candidate compound or nucleic acid; and
(c) measuring an activity of said target.
The target may be a messenger RNA molecule and said activity may be translation of said messenger RNA. The target may be a messenger RNA molecule and said activity may be transcription of a gene encoding said messenger RNA. The target may be a gene and said activity may be transcription of said gene. The target may be a nontranslated RNA and said activity may be processing or folding of said nontranslated RNA or assembly of said nontranslated RNA into a protein/RNA complex. The target gene or RNA may encode a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 299-469.
Another embodiment of the present invention is a compound or nucleic acid identified using the methods of the preceding paragraph.
Another embodiment of the present invention is a method for identifying a compound which reduces the activity or level of a gene product required for proliferation of a microorganism, wherein the activity or expression of said gene product is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127, said method comprising the steps of:
(a) expressing a sublethal level of an antisense nucleic acid complementary to a nucleic acid encoding said gene product in a cell to reduce the activity or amount of said gene product in said cell, thereby producing a sensitized cell;
(b) contacting said sensitized cell with a compound; and
(c) determining whether said compound inhibits the growth of said sensitized cell.
The determining step may comprise determining whether said compound inhibits the growth of said sensitized cell to a greater extent than said compound inhibits the growth of a nonsensitized cell. The cell may be selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells. The cell may be a Gram negative bacterium. The cell may be an E. coli cell. The cell may be from an organism selected from the group consisting of Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Saccharomyces cerevisae, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum, and Yersinia pestis or any species falling within the genera of any of the above species. The antisense nucleic acid may be transcribed from an inducible promoter. The method may further comprise the step of contacting said cell with a concentration of inducer which induces said antisense nucleic acid to a sublethal level. Growth inhibition may be measured by monitoring optical density of a culture growth solution. The gene product may be a polypeptide. The polypeptide may comprise a sequence selected from the group consisting of SEQ ID NOs.: 299-469. The gene product may be an RNA.
Another embodiment of the present invention is a compound identified using the methods of the preceding paragraph.
Another embodiment of the present invention is a method for inhibiting cellular proliferation comprising introducing a compound with activity against a gene whose activity or expression is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127 or a compound with activity against the product of said gene into a population of cells expressing said gene. The compound may be an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127, or a proliferation-inhibiting portion thereof. The proliferation inhibiting portion of one of SEQ ID NOs.: 1-127 may be a fragment comprising at least 10, at least 20, at least 25, at least 30, at least 50 or more than 51 consecutive bases of one of SEQ ID NOs.: 1-127. The population may be a population selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells. The population may be a population of Gram negative bacteria. The population may be a population of E. coli cells. The population may be a population selected from the group consisting of Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa,Saccharomyces cerevisae, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum, and Yersinia pestis cells or cells from any species falling within the genera of any of the above species. The gene may encode a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 299-469.
Another embodiment of the present invention is a preparation comprising an effective concentration of an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127, or a proliferation-inhibiting portion thereof in a pharmaceutically acceptable carrier. The proliferation-inhibiting portion of one of SEQ ID NOs.: 1-127 may comprise at least 10, at least 20, at least 25, at least 30, at least 50 or more than 50 consecutive bases of one of SEQ ID NOs.: 1-127.
Another embodiment of the present invention is a method for inhibiting the activity or expression of a gene in an operon required for proliferation wherein the activity or expression of at least one gene in said operon is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127, said method comprising contacting a cell in a cell population with an antisense nucleic acid comprising at least a proliferation-inhibiting portion of said operon. The antisense nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs.: 1-127 or a proliferation inhibiting portion thereof. The method of claim 68, wherein said cell is contacted with said antisense nucleic acid by introducing a plasmid which expresses said antisense nucleic acid into said cell population. The cell may be contacted with said antisense nucleic acid by introducing a phage which expresses said antisense nucleic acid into said cell population. The cell may be contacted with said antisense nucleic acid by expressing said antisense nucleic acid from the chromosome of cells in said cell population. The cell may be contacted with said antisense nucleic acid by introducing a promoter adjacent to a chromosomal copy of said antisense nucleic acid such that said promoter directs the synthesis of said antisense nucleic acid. The cell may be contacted with said antisense nucleic acid by introducing a retron which expresses said antisense nucleic acid into said cell population. The cell may be contacted with said antisense nucleic acid by introducing a ribozyme into said cell-population, wherein a binding portion of said ribozyme is complementary to said antisense oligonucleotide. The cell may be contacted with said antisense nucleic acid by introducing a liposome comprising said antisense oligonucleotide into said cell. The cell may be contacted with said antisense nucleic acid by electroporation of said antisense nucleic acid. The antisense nucleic acid may be a fragment comprising at least 10, at least 20, at least 25, at least 30, at least 50 or more than 50 consecutive bases of one of SEQ ID NOs.: 1-127. The antisense nucleic acid may be an oligonucleotide.
Another embodiment of the present invention is a method for identifying a gene which is required for proliferation of a microorganism comprising:
(a) contacting a microorganism other than E. coli with a nucleic acid selected from the group consisting of SEQ ID NOs.: 1-127;
(b) determining whether said nucleic acid inhibits proliferation of said microorganism; and
(c) identifying the gene in said microorganism which is inhibited by said nucleic acid.
The microorganism may be a Gram negative bacterium. The microorganism may be selected from the group consisting of Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Saccharomyces cerevisae, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum, and Yersinia pestis or any species falling within the genera of any of the above species. The method may further comprise introducing said nucleic acid into a vector functional in said microorganism prior to introducing said inhibitory nucleic acid into said microorganism.
Another embodiment of the present invention is a method for identifying a compound having the ability to inhibit proliferation of a microorganism comprising:
(a) identifying in a first microorganism a homolog of a gene or gene product present in a second microorganism which is different than said first microorganism, wherein the activity or level of said gene or gene product is inhibited by a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs. 1-127;
(b) identifying an inhibitory nucleic acid sequence which inhibits the activity of said homolog in said first microorganism;
(c) contacting said first microorganism with a sublethal level of said inhibitory nucleic acid, thus sensitizing said first microorganism;
(d) contacting the sensitized microorganism of step (c) with a compound; and
(e) determining whether said compound inhibits proliferation of said sensitized microorganism.
The determining step may comprise determining whether said compound inhibits proliferation of said sensitized microorganism to a greater extent than said compound inhibits proliferation of a nonsensitized microorganism. Step (a) may comprise identifying a homologous nucleic acid to a gene or gene product whose activity or level is inhibited by a nucleic acid selected from the group consisting of SEQ ID NOs. 1-127 or a nucleic acid encoding a homologous polypeptide to a polypeptide whose activity or level is inhibited by a nucleic acid selected from the group consisting of SEQ ID NOs. 1-127 by using an algorithm selected from the group consisting of BLASTN version 2.0 with the default parameters and FASTA version 3.0t78 algorithm with the default parameters to identify said homologous nucleic acid or said nucleic acid encoding a homologous polypeptide in a database. Step (a) may comprise identifying a homologous nucleic acid or a nucleic acid encoding a homologous polypeptide by identifying nucleic acids which hybridize to said first gene. Step (a) may comprise expressing a nucleic acid selected from the group consisting of SEQ ID NOs. 1-127 in said microorganism. The inhibitory nucleic acid may be an antisense nucleic acid. The inhibitory nucleic acid may comprise an antisense nucleic acid to a portion of said homolog. The inhibitory nucleic acid may comprise an antisense nucleic acid to a portion of the operon encoding said homolog. The step of contacting the first microorganism with a sublethal level of said inhibitory nucleic acid may comprise directly contacting said microorganism with said inhibitory nucleic acid. The step of contacting the first microorganism with a sublethal level of said inhibitory nucleic acid may comprise expressing an antisense nucleic acid to said homolog in said microorganism. The gene product may comprise a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 299-469.
Another embodiment of the present invention is a compound identified using the method of the preceding paragraph.
Another embodiment of the present invention is a method of identifying a compound having the ability to inhibit proliferation comprising:
(a) contacting a microorganism other than E. coli with a sublethal level of a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs. 1-127 or a portion thereof which inhibits the proliferation of E. coli, thus sensitizing said microorganism;
(b) contacting the sensitized microorganism of step (a) with a compound; and
(c) determining whether said compound inhibits proliferation of said sensitized microorganism.
The determining step may comprise determining whether said compound inhibits proliferation of said sensitized microorganism to a greater extent than said compound inhibits proliferation of a nonsensitized microorganism.
Another embodiment of the present invention is a compound identified using the methods of the preceding paragraph.
Another embodiment of the present invention is a method for identifying a compound having activity against a biological pathway required for proliferation comprising:
(a) sensitizing a cell by expressing a sublethal level of an antisense nucleic acid complementary to a nucleic acid encoding a gene product required for proliferation, wherein the activity or expression of said gene product is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-27, in said cell to reduce the activity or amount of said gene product;
(b) contacting the sensitized cell with a compound; and
(c) determining whether said compound inhibits the growth of said sensitized cell.
The determining step may comprise determining whether said compound inhibits the growth of said sensitized cell to a greater extent than said compound inhibits the growth of a nonsensitized cell. The cell may be selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells. The cell may be a Gram negative bacterium. The Gram negative bacterium may be E. coli. The cell may be selected from the group consisting of Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Saccharomyces cerevisae, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum, and Yersinia pestis or any species falling within the genera of any of the above species. The antisense nucleic acid may be transcribed from an inducible promoter. The method may further comprise contacting the cell with an agent which induces expression of said antisense nucleic acid from said inducible promoter, wherein said antisense nucleic acid is expressed at a sublethal level. The inhibition of proliferation may be measured by monitoring the optical density of a liquid culture. The gene product may comprise a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 299-469.
Another embodiment of the present invention is a compound identified using the methods of the preceding paragraph.
Another embodiment of the present invention is a method for identifying a compound having the ability to inhibit cellular proliferation comprising:
(a) contacting a cell with an agent which reduces the activity or level of a gene product required for proliferation of said cell, wherein said gene product is a gene product whose activity or expression is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127;
(b) contacting said cell with a compound; and
(c) determining whether said compound reduces proliferation of said contacted cell.
The determining step may comprise determining whether said compound reduces proliferation of said contacted cell to a greater extent than said compound reduces proliferation of cells which have not been contacted with said agent. The agent which reduces the activity or level of a gene product required for proliferation of said cell may comprise an antisense nucleic acid to a gene or operon required for proliferation. The agent which reduces the activity or level of a gene product required for proliferation of said cell may comprise a compound known to inhibit growth or proliferation of a microorganism. The cell may contain a mutation which reduces the activity or level of said gene product required for proliferation of said cell. The mutation may be a temperature sensitive mutation. The gene product may comprise a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 299-469.
Another embodiment of the present invention is a compound identified using the method of the preceding paragraph.
Another embodiment of the present invention is a method for identifying the biological pathway in which a proliferation-required gene or its gene product lies, wherein said gene or gene product comprises a gene or gene product whose activity or expression is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127, said method comprising:
(a) expressing a sublethal level of an antisense nucleic acid which inhibits the activity of said proliferation-required gene or gene product in a cell;
(b) contacting said cell with a compound known to inhibit growth or proliferation of a microorganism, wherein the biological pathway on which said compound acts is known; and
(c) determining whether said cell is sensitive to said compound.
The determining step may comprise determining whether said cell has a substantially greater sensitivity to said compound than a cell which does not express said sublethal level of said antisense nucleic acid and wherein said gene or gene product lies in the same pathway on which said compound acts if said cell expressing said sublethal level of said antisense nucleic acid has a substantially greater sensitivity to said compound than said cell which does not express said sublethal level of said antisense nucleic acid. The gene product may comprise a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 299-469.
Another embodiment of the present invention is a method for determining the biological pathway on which a test compound acts comprising:
(a) expressing a sublethal level of an antisense nucleic acid complementary to a proliferation-required nucleic acid in a cell, wherein the activity or expression of said proliferation-required nucleic acid is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127 and wherein the biological pathway in which said proliferation-required nucleic acid or a protein encoded by said proliferation-required polypeptide lies is known,
(b) contacting said cell with said test compound; and
(c) determining whether said cell is sensitive to said test compound.
The determining step may comprise determining whether said cell has a substantially greater sensitivity to said test compound than a cell which does not express said sublethal level of said antisense nucleic acid. The method may further comprise:
(d) expressing a sublethal level of a second antisense nucleic acid complementary to a second proliferation-required nucleic acid in a second cell, wherein said second proliferation-required nucleic acid is in a different biological pathway than said proliferation-required nucleic acid in step (a); and
(e) determining whether said second cell does not have a substantially greater sensitivity to said test compound than a cell which does not express said sublethal level of said second antisense nucleic acid, wherein said test compound is specific for the biological pathway against which the antisense nucleic acid of step (a) acts if said second cell does not have substantially greater sensitivity to said test compound.
Another embodiment of the present invention is a purified or isolated nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127.
Another embodiment of the present invention is a compound which interacts with a gene or gene product whose activity or expression is inhibited by an antisense nucleic acid comprising one of SEQ ID NOs.: 1-127 to inhibit proliferation.
Another embodiment of the present invention is a compound which interacts with a polypeptide whose expression is inhibited by an antisense nucleic acid comprising one of SEQ ID NOs.: 1-127 to inhibit proliferation.
Another embodiment of the present invention is a method for manufacturing an antibiotic comprising the steps of screening one or more candidate compounds to identify a compound that reduces the activity or level of a gene product required for proliferation, said gene product comprising a gene product whose activity or expression is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-27 and manufacturing the compound so identified. The screening step may comprise performing any one of the methods of identifying a compound described above.
Another embodiment of the present invention is a method for inhibiting proliferation of a microorganism in a subject comprising administering a compound that reduces the activity or level of a gene product required for proliferation of said microorganism, said gene product comprising a gene product whose activity or expression is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 1-127 to said subject. The method of subject may be selected from the group consisting of vertebrates, mammals, avians, and human beings. The gene product may comprise a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 299-469.